Abstract Drug-resistant pathogens are causing a human health crisis. The overall goal of this Program is to resolve the bottleneck in the antimicrobial pipeline by developing an effective platform to discover novel compounds produced by an untapped source of chemical diversity, uncultured bacteria. The goal of this component project is to identify the mode of action of known antimicrobials with unknown target; and compounds produced by uncultured bacteria. Natural products have been all but abandoned by the Industry due to overmining of culturable bacteria and diminishing returns, but surprising discoveries keep coming from this group of compounds, both new and old. We discovered that acyldepsipeptide is capable of killing dormant persisters and sterilizes an incurable biofilm infection activating proteolysis. Novel species-selective compounds have been described ? cyclomarin corrupts the essential ClpP1P2C1 protease of M. tuberculosis and lassomycin, which we recently isolated from an uncultured bacterium, inhibits this protease and simultaneously activates its ATPase, resulting in killing dormant cells. Aspergillomarasmine, a rediscovered old compound, inhibits metal ?-lactamases which are insensitive to available therapies. Finally, we recently discovered teixobactin, a novel inhibitor of peptidoglycan synthesis from Elephtheria terrae, an uncultured ?-proteobacterium. The compound binds to both lipid II and lipid III and is essentially free of resistance. We will undertake a large effort to identify the MOA of 500 known compounds with unknown targets. Their transcriptomes (Project II) will indicate specificity of action, and candidates will be evaluated in this project. Resistant mutants will be obtained, and sequencing of the target will provide independent confirmation. The MOA of a selected number of attractive candidates for development will be studied in more detail. This will considerably enrich our knowledge of what evolution determined to be a good target and facilitate the determination of MOA of new compounds produced by uncultured bacteria. Laborious chemical dereplication is a bottleneck that severely limits the rate of discovery of natural products. Rapid detection of the likely MOA of compounds present in extracts and fractions by transcriptome analysis (Project II) will resolve this bottleneck. Candidate leads from both old known compounds and the ones we discover from uncultured bacteria will be characterized in vitro and in vivo for potency, toxicity, and efficacy. For compounds with bactericidal activity, we will examine in detail activity against dormant persister cells and biofilms. Compounds that show good efficacy will become validated leads for advanced drug development. Unleashing the production potential of uncultured bacteria is likely to tip the balance in the standoff with pathogens in our favor. Determining the MOA of candidate compounds, together with their in vitro and in vivo properties will validate this drug discovery platform. The leads we characterize will become candidates for drug development.